Many cell types release molecules packaged in membrane bound secretory vesicles or granules by exocytosis. This mechanism mediates release of neurotransmitters, hormones as well as many other compounds including histamine from mast cells or cytotoxic proteins from eosinophilic granulocytes. The readily releasable vesicles of neurons and neuroendocrine cells are awaiting stimulation in a state tethered to their release site at the plasma membrane. It remains unclear what molecular mechanism forms a physical link between a vesicle and its target membrane. This knowledge gap is due to the lack of a technique that can physically measure tethering interaction of a vesicle with the plasma membrane and quantitatively probe the forces involved in this interaction. We propose to develop a technology that fills this gap allowing characterization of vesicle-plasma membrane tethering interactions. PC12 cell plasma membrane sheets with tethered vesicles exposed on the cytoplasmic side of the membrane sheet will be used as the preparation. A method will be developed to bind an AFM tip firmly bound to a granule tethered to the plasma membrane by the physiological tethering components. For precise monitoring of the vesicle movement perpendicular to the plasma membrane the AFM is mounted on a fluorescence microscope with TIRF capability. Tether dissociation events can thereby be distinguished from a dissociation of the AFM tip from the vesicle and provide detailed information on the tethering interactions. Tether dissociation kinetics and landscape will be characterized as a function of applied force. The properties of newly formed tethers will be compared with those of pre-existing tethers. This technology development will provide the foundation for studies using pharmacological and molecular manipulations to elucidate the molecular mechanism of tethering and for comparison with experiments on reconstituted systems. If successful, this enabling technology will considerably advance our understanding of tethering and fusion, which is a ubiquitous mechanism in exocytosis and intracellular trafficking. PUBLIC HEALTH RELEVANCE: In health and disease, different cell types release various molecules in response to specific needs of the organism from membrane bound vesicles. For this function, the vesicles must be tethered at the release site and release of hormones, neurotransmitters and mediators of the immune system is impaired when tethering is disrupted. This project develops a technology capable of measuring the molecular forces that hold vesicle at the right site in the cell to advance our understanding of this intricate biological molecular tethering apparatus.